Stable troponin subunits and complexes

ABSTRACT

Stable troponin subunits and complexes and methods for their preparation are described. Among other uses, these subunits and complexes are useful as antigens for the preparation of antibodies, and as controls and calibrators for troponin assays. One complex comprises a modified human cardiac troponin I together with human cardiac troponin T and human cardiac troponin C. Another complex comprises a modified human cardiac troponin I with human cardiac troponin C.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending and commonly owned application Ser. No. 08/862,613, filed May 23, 1997, now abandoned.

FIELD OF THE INVENTION

This invention relates to recombinantly-expressed human cardiac troponin subunits I, C, and T, recombinant human cardiac troponin complexes made in vitro therefrom, and methods for the production and purification of the subunits and the preparation of the complexes.

BACKGROUND OF THE INVENTION

Early and accurate assessment of suspected acute myocardial infarction is critically dependent on the sensitive and specific detection and quantitation in blood, serum or plasma of released cardiac muscle intracellular components in order to distinguish a potentially lethal event in need of emergency measures from non-life threatening conditions such as angina and non-cardiac chest pain such as dyspepsia. Early electrocardiographic changes are not adequately specific, and the medical profession has come to rely on serum biochemical markers of cardiac tissue necrosis for early diagnosis. Initially, the serum markers creatine kinase (CK) and specifically the cardiac CK-MB isoform were used, and subsequently myoglobin as a more sensitive early indicator of cardiac damage. More recently, the highly sensitive markers cardiac troponin subunits I and T have come to be preferred for their extraordinarily high specificity for identifying myocardial damage. These tests, along with other markers of skeletal muscle necrosis, provide a high degree of diagnostic accuracy. If performed in the emergency room, an early and accurate diagnosis of myocardial damage offers great advantage to a suspected heart attack victim.

Diagnostic tests employing highly sensitive cardiac markers are described, for example, in U.S. Pat. Nos. 5,604,105 and 5,290,678. These procedures offer the rapidity of diagnosing myocardial infarction in the emergency room setting and offer significant medical benefit for patients.

Though numerous diagnostic assays for troponin subunits, principally I and T, exist, for example the troponin I Stratus(R) test from Dade International, Inc., the Opus(R) test from Behring, and the Access(R) test from Sanofi, there is no troponin subunit or complex preparation of sufficient quality and stability to employ as a standard for calibration of assays nor to use as a universally acceptable consensus standard across the industry. In order to maintain the conformational structure of troponin I, it must be complexed with troponin C. Troponin preparations from one source and for one particular assay format may not be useable in another, thus it is not currently possible to use the same standard across tests from different manufacturers. The lack of a industry-accepted standard limits the establishment of industry guidelines for test criteria as well as comparison of results throughout the world to help establish normal and abnormal value ranges.

Numerous troponin preparations from both natural and recombinant sources have been proposed. Previously described methods for the purification of troponin subunits from cardiac tissue (Tsukui et al., 1973, J. Biochem., v. 73, pp. 1119-1121; Cummins et al., 1978, Biochem. J., v. 171, pp. 251-259; Syska et al., 1974, FEBS Letts., v. 40, pp. 253-257) yielded preparations which were unstable and subject to considerable degradation on storage. A troponin complex prepared from cardiac tissue or from the combination of isolated troponin subunits prepared from cardiac tissue has been described (EP 0 743 522 A1). DE4405249 (U.S. Pat. No. 5,583,200) describes a stabilized troponin I or T also containing troponin C, with stability of several days in the cold. Use of a combination of four protease inhibitors in U.S. Pat. No. 5,560,937 overcame the degradation and stability issues inherent in tissue-derived troponin I, but did not obviate the need for human heart tissue. Unfortunately, human tissue is a potential source of infection, including HIV and hepatitis, to workers during troponin purification, and human heart tissue is of increasing scarcity especially with the successful use of early diagnostic tests for heart attack responsible for decreasing mortality from this disease. Troponin standards prepared by recombinant means offer a less costly and less hazardous alternative to that from heart tissue, but may still suffer from a lack of stability incompatible with industry demands.

Recombinant troponin subunits and complexes have been described. Armour et al. (1993, Gene, v. 131, pp. 287-292) cloned human cardiac troponin I in a bacterial system and expressed the gene product both as a beta-gal fusion product, and as an unfused product. Al-Hillawi et al. (1994, Eur. J. Biochem., v. 225, pp. 1195-1201) expressed human cardiac troponin I and troponin C in E. coli, using two codon changes in the cDNA of the former to overcome difficulties in expressing the human product in bacteria. Malnic and Reinach (1994, Eur. J. Biochem., v. 222, pp. 49-54) produced a recombinant complex in vivo by cloning all three chicken skeletal muscle troponin subunits into an expression plasmid. The troponin complex formed within the bacterium. Fujita-Becker et al. (1993, J. Biochem., v. 114, pp. 438-444) described the reconstitution of rabbit skeletal troponin complex from recombinant subunits expressed in E. coli. None of these recombinant products has been demonstrated to have adequate stability for use as a diagnostic test standard or calibrator. A recombinant human cardiac troponin complex formed in vitro from recombinant human cardiac troponin I, recombinant human cardiac troponin T, and recombinant human cardiac troponin C has not been described previously, nor has a complex formed of recombinant human cardiac troponin I and recombinant human cardiac troponin C.

Thus, there is a need for troponin complexes which meet the requirements of a stable material of safe origin and economical preparation that may be used as controls or calibrators across troponin assays. It has now been discovered that human cardiac troponin complexes prepared from recombinant subunits offer, among other advantages, superior stability and utility among troponin assays to be suitable for use as a universal standard.

SUMMARY OF THE INVENTION

The present invention relates to a recombinant human cardiac troponin complex comprising a recombinant modified human cardiac troponin I, recombinant human cardiac troponin C, and recombinant human cardiac troponin T, the complex formed in vitro from the individual recombinant troponin subunits. The invention also comprises a complex formed of recombinant modified human cardiac troponin I and recombinant human cardiac troponin C. The present invention also relates to a novel recombinant human cardiac troponin I subunit with a modified cDNA and protein sequence with improved expression in E. coli. The troponin complexes prepared in vitro from the recombinantly expressed subunits are of higher stability than previously reported troponin complexes prepared from recombinant methods or from troponin subunits isolated from cardiac tissue. They can be used as controls or calibrators among different troponin assay procedures. In contrast, previously known troponins cannot be so employed.

The subunits and complexes are also useful as antigens to prepare antibodies useful for a variety of purposes.

The troponin subunits are prepared by recombinant techniques, expressed and optionally purified individually before admixture to prepare the complexes. The complex may be purified by standard procedures after they are formed. Recombinant expression of the troponin subunits can be optimized. Troponin I is engineered as a recombinant product with an additional N-terminal portion of from about 4 to about 12, preferably 5 to 8, amino acids. It has been observed that the additional amino acids increase the expression of modified troponin I in E. coli. Troponin T has been expressed at high levels by altering arginine codons in the cDNA to be compatible with the bacterial expression system, without altering the amino acid sequence of the subunit. Recombinant troponin C is expressed with the native amino acid sequence.

Modified Troponin I as used herein refers to Troponin I with additional N-terminal acids to increase the expression of the desired product. It is not essential that the amino acids be identical with or in the same order as shown in the figure.

The subunits are prepared separately before being brought together in vitro under selected conditions to form the troponin complexes. Conditions are selected to promote complex formation, including the use of an alkaline earth salt or salts and, if desired, a detergent for the complex of troponins C, I and T. One or more alkaline earth salts, and a chaotropic agent, may be employed in the preparation of the complex of troponin C and I. Calcium chloride and magnesium chloride are preferred as alkaline earth salts. As the detergent, sodium dodecyl sulfate is preferred. Urea is the preferred chaotropic agent.

It is an objective of the present invention to provide a recombinant human cardiac troponin I protein of SEQ ID NO:5 which has an additional N-terminus of about 6 amino acids and other codon changes which facilitates the recombinant expression of the protein. It is a further objective of this invention to provide a recombinant human cardiac troponin T protein produced from a cDNA sequence set forth in SEQ ID NO:4 which has certain codon changes in the cDNA but no change in protein sequence compared to native human cardiac troponin T.

According to the present invention, a method is provided for the preparation of human cardiac troponin complexes prepared from recombinant modified troponin I, recombinant troponin C, and recombinant troponin T. The method comprises the steps of: 1) expressing the troponin subunits recombinantly; 2) optionally purifying the separate subunits; 3) admixing the subunits preferably in the presence of at least one alkaline earth salt or salts and, if desired, a detergent, to form the troponin complex. The use of a detergent increases the yield and purity of the products. It is a further object of the invention to provide a method for the preparation of a human cardiac troponin complex prepared from recombinant modified troponin I and recombinant troponin C, comprising the steps of: 1) expressing the troponin subunits recombinantly; 2) admixing the subunits preferably in the presence of an alkaline earth salt or salts and a chaotropic agent to form the troponin complex. Purification is preferably effected before formation of a complex. The present invention contemplates modifications to the DNA sequence of the troponin subunits to facilitate their expression in their respective recombinant systems.

The recombinant troponin complexes according to the present invention have the purity and stability characteristics to enable utility as assay controls and calibrators for use in the diagnostics industry for the manufacture, quality control, and calibration of troponin assays. The troponin complexes of the present invention show superior stability compared to existing troponin complexes, and may be utilized as controls among different troponin assay procedures, in contrast to existing troponin controls which cannot be used except in the specific assays for which they were designed.

It is a further advantage of the present invention that production of the recombinant human cardiac troponin complexes is independent of the availability of human cardiac tissue. Working with human tissues presents an inherent risk of infection by any number of diseases including HIV and hepatitis. In addition, the quality of tissue may be unknown resulting in the production of an inferior product. Heart tissue from elderly individuals may be hypertrophic and as a result have a low muscle mass and thus not provide a satisfactory quantity or quality of troponin. Human heart tissue is prohibitively costly as a long-term, consistent source of troponin to meet the future demands of the rapidly expanding field of emergency cardiac diagnostics.

Another advantage of the present invention is the ease of purification compared to products isolated from human cardiac tissue. Consequently, the authenticity and purity of each subunit can be simply determined prior to the preparation of the complexes. The lack of uniformity of tissue, presence of degradation or autolysis products, etc., are obviated by use of the individual recombinant subunits rather than products isolated from natural sources.

A further advantage of the present invention is that it provides a novel recombinant modified human cardiac troponin I that may be used as an assay control or calibrator.

A still further advantage is that the invention provides recombinant troponin subunits of sufficient purity and quantity to permit their use as controls or calibrators and also to serve as substrates from which to produce stable troponin complexes that meet the purity and stability requirements to be used as an assay standard or calibrator.

These and other advantages of the recombinant human cardiac troponin complexes and its subunits in the present invention will be apparent upon consideration of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the DNA (SEQ ID NO: 1) and protein (SEQ ID NO: 5) sequences of the recombinant modified human cardiac troponin I of the present invention. The box points out the six amino acid residues added to the N-terminus of human cardiac troponin I. The original bases (SEQ ID NO: 3, including the additional N-terminal amino acids) are shown above the altered sequences.

FIG. 2 represents the DNA (SEQ ID NO: 4) and protein (SEQ ID NO: 6) sequences of the recombinant human cardiac troponin T of the present invention. The codons which replace the natural codon is underlined, and the natural codon is shown above. The native troponin T DNA sequence is shown in SEQ ID NO: 2.

DETAILED DESCRIPTION OF THE INVENTION

Measurement in circulation of the cardiac muscle-associated protein troponin and, particularly, its components I and T, has proven to be an early and specific indicator of suspected acute myocardial infarction. As such, numerous methods for rapidly and accurately detecting troponin and its subunits in blood have been and are being developed for diagnosing heart attack in an emergency situation, and countless lives have been and will be saved as a result. However, in order to develop accurate and dependable diagnostic assays and to calibrate them, the availability of stable, high-quality human cardiac troponin controls is critical for quality control and testing purposes. This invention provides pure, stable, recombinant human cardiac troponin complexes to meet the needs of the industry. A troponin complex may be formed in vitro from the admixture of recombinantly expressed human cardiac troponin I, troponin T and troponin C. Another troponin complex may be formed from the admixture of recombinantly expressed human cardiac troponin I and C.

Troponin I may be engineered as a recombinant product with about 6 additional amino acid N-terminal residues (see FIG. 1; SEQ ID NO:5) to increase its expression in E. coli. In addition, certain codon changes have been made in the cDNA sequence to optimize expression of the protein in E. coli, without change in the amino acid sequence. The modified recombinant troponin I may be isolated from the bacterial host and purified by standard protein purification methods following details illustrated in the examples below. The purified modified troponin I is then be employed as an antigen or to prepare the recombinant troponin complexes such as that of troponin I, troponin C and troponin T, and that of troponin I and troponin C. Troponin C stabilizes the conformational structure of troponin I. In addition, the recombinant, modified human cardiac troponin I has utility itself as an improved control or calibrator.

Recombinant troponin T has been expressed at high levels by altering arginine codons in the cDNA (see FIG. 2; SEQ ID NO:4) to be compatible with the bacterial expression system, without altering the amino acid sequence of the subunits. The recombinant troponin T is isolated from the bacterial host and, optionally, purified by standard protein purification methods following details in the example below. The recombinant troponin T produced by this method has utility itself as an improved control or calibrator.

The troponin C is expressed in E. coli in its native amino acid sequence, then isolated from the bacterial host and optionally purified by standard protein purification methods following details illustrated in the examples below.

Typically, the subunits are prepared separately and optimally purified before being brought together in vitro under selected conditions to form the complex of troponin I, T and C. Conditions are selected to promote complex formation, including the use of an alkaline earth salt or salts and, if desired, a detergent. As the alkaline earth salts, calcium chloride and magnesium chloride are preferred. As the detergent, sodium dodecyl sulfate is preferred. For the complex of troponin I and C, conditions are selected to promote complex formation including the use of an alkaline earth salt or salts and a chaotropic agent. As the alkaline earth salts, calcium chloride and magnesium chloride are preferred. As the chaotropic agent, urea is preferred.

As will be evident from the examples presented below, the recombinant troponin complexes according to the present invention have purity and stability characteristics to enable their utility as assay controls and calibrators for use in the diagnostics industry for the manufacture, quality control, and calibration of troponin assays. The troponin complexes of the present invention shows superior stability compared to known troponin complexes, and are shown to be detected utilizing different troponin assay methods and instruments, in contrast to existing troponin controls which cannot be used as a standard outside of the specific assay for which they were designed.

The preparation of the recombinant troponin subunits, their purification, and preparation of the complexes are illustrated in the following non-limiting examples, which also demonstrate their stability and utility in various assays.

EXAMPLE 1 Expression and purification of modified human cardiac troponin I

Human cardiac troponin I cDNA was cloned by the polymerase chain reaction (PCR) using primers designed from published cardiac troponin I cDNA sequence (Vallins et al., 1990, FEBS Letters, vol. 270, pp. 57-61, the full disclosure of which is incorporated herein by reference). Since the expression of human cardiac troponin I was very low in E. coli if the original cDNA sequence was used (Armour et al., 1993, Gene, vol. 131, pp. 287-292), both N-terminus addition and codon alteration were performed to increase the yield. The altered human cardiac troponin I cDNA sequence (FIG. 1; SEQ ID NO: 1) was confirmed by DNA sequencing and cloned into plasmid pET21 (Novagen). E. coli BL21 (DE3) cells (Novagen) were transformed with the resulting construct and the protein produced from this expression vehicle analyzed using SDS-PAGE. This E. coli strain has been deposited and has ATCC number.

For purification, the following reagents were used: lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 20 mM 2-mercaptoethanol, 100 mM sodium chloride, pH 8.0); 2M urea-lysis buffer (2M urea in lysis buffer; 8M urea-lysis buffer (8M urea in lysis buffer); dialysis buffer (20 mM Tris-HCl, 100 mM sodium chloride, 10 mM 2-mercaptoethanol, pH 7.5); eluting buffer (20 mM Tris-HCl, 800 mM sodium chloride, 10 mM 2-mercaptoethanol, pH 7.5); final buffer (20 mM Tris-HCl, 500 mM sodium chloride, 60 mM 2-mercaptoethanol, pH 7.5).

The following sequential steps were carried out for the purification of the modified human recombinant troponin I protein. Recombinant bacterial cells were collected by centrifugation of the culture medium. Cells were resuspended in lysis buffer at the ratio of 50 ml lysis buffer per liter equivalent of original culture medium. Lysozyme (10 mg/ml) was added at 1 ml lysozyme per liter of culture medium. The resulting mixture was then shaken at room temperature for 30 minutes. Triton X-100 (10%) was added at 5 ml per liter of culture medium, followed by PMSF (0.1M) at 0.5 ml per liter of medium. The suspension was incubated at 37° C. for 15 minutes. The vessel containing the suspension was submerged in dry ice/ethanol to rapidly freeze the cell suspension. The frozen suspension was then thawed in a 37° C. water bath. The thawed cell suspension was sonicated using a Vibra Cell™ sonicator at 2° C.-8° C. to shear the DNA. The suspension was centrifuged at 20,000 g for 20 min at 2° C.-8° C. and the supernatant was discarded. Fifty ml of 2M urea-lysis buffer and 10 ml of 10% Triton X-100 was added per liter equivalent of original culture medium; the pellet was homogenized and stirred at 4° C. for 15 min. The mixture was then centrifuged at 20,000 g for 20 min at 2° C.-8° C. and the supernatant discarded. 8M urea-lysis buffer (50 ml of urea-lysis buffer per liter cell culture) was added, and the mixture homogenized then stirred for 30 min at room temperature. After centrifugation at 20,000 g at 2° C.-8° C. for 20 min. the pellet is discarded. The supernatant is dialyzed in dialysis buffer overnight, and if required, the dialysate was centrifuged to remove any particulate matter.

The sample was loaded onto a previously equilibrated CM-Sephadex column (for example, a 2.5×20 cm; 100 ml bed volume for 2 L of original culture medium). The column was washed with 4 bed volumes of the dialysis buffer, and eluted with a linear gradient of 3.5 bed volumes of 0-800 mM NaCl (100% dialysis buffer changed over to 100% eluting buffer). The fractions were checked by 15% SDS-PAGE and those containing the troponin I were pooled then dialyzed against final buffer. The dialysate was centrifuged at 20,000 g for 20 min and the supernatant filtered through two layers of Miracloth. In order to remove endotoxin from the protein preparation and form a product useful as an antigen, following Triton X-114 phase separation procedures are performed. Triton X-114 (100%) was added at 1 ml per 100 ml of the supernatant and stirred at 4° C. for 30 min. The sample was incubated at 37° C. for 10 min., then centrifuged at 20,000 g for 5 min at 30° C. using 40-ml centrifuge tubes. After removing the upper aqueous phase with care so as not to aspirate the detergent phase, the aqueous phases were combined. The addition and centrifugation steps with Triton X-114 were repeated two more times. The aqueous phase was dialyzed overnight against the final buffer (100 ml sample per liter of the final buffer).

Final determination of protein concentration was performed using the Bradford Assay, using Bradford BSA as the standard, and final purity using a 15% SDS-PAGE. The purified recombinant human cardiac troponin I was stored at -20° C.

EXAMPLE 2 Expression and purification of troponin T

Human cardiac troponin T was cloned and expressed as previously described (Hu et al., 1996, Protein Expression and Purification, vol. 7, pp. 289-293, the full disclosure of which is incorporated herein by reference). According to this method, the codons of two consecutive pairs of AGG codons in the human cardiac troponin T cDNA were replaced in order to achieve a high level of expression in E. coli. The replacements were with synonymous codons thus the protein produced from this expression vehicle was not altered (see FIG. 2; SEQ ID NO:6). A 40-fold increased expression was achieved. This E. coli strain has been deposited and has ATCC number.

For purification of recombinant troponin T, the following reagents were used: lysis buffer: (50 mM Tris-HCl, 1 mM EDTA, 20 mM 2-mercaptoethanol, 100 mM sodium chloride, pH 8.0); dialysis buffer 1 (50 mM Tris-HCl, 500 mM potassium chloride, 5 mM 2-mercaptoethanol, 1 mM EDTA, pH 8.0); dialysis buffer II (50 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 8.0); eluting buffer (50 mM Tris-HCl, 500 mM sodium chloride, 5 mM 2-mercaptoethanol, pH 8); storage buffer (50 mM Tris-HCl, 100 mM sodium chloride, 5 mM 2-mercaptoethanol, pH 7.5).

Purification of recombinant troponin T was accomplished as follows. Recombinant bacterial cells were collected by centrifugation of the cell culture medium. Cells were resuspend in lysis buffer at the ratio of 50 ml lysis buffer per liter equivalent of culture medium. Lysozyme (10 mg/ml) was added at 1 ml lysozyme per liter of culture medium. For antigen preparation, the resulting mixture was then shaken at room temperature for 30 minutes. Triton X-100 (10%) was added at 5 ml per liter of original culture medium, followed by PMSF (0.1M) at 0.5 ml of PMSF per liter of medium. The suspension was incubated at 37° C. for 15 minutes. The vessel containing the suspension was submerged into dry ice/ethanol to rapidly freeze the cell suspension. The frozen suspension was then thawed in a 37° C. water bath. The thawed cell suspension was sonicated using a Vibra Cell™ sonicator at 2° C.-8° C. to shear the DNA. The suspension was centrifuged at 20,000 g for 20 min at 2° C.-8° C. and the pellet was discarded. The supernatant was dialyzed in dialysis buffer I overnight, and if required, the dialysate was centrifuged to remove any particular matter.

The troponin T in the supernatant was precipitated with 30-50% ammonium sulfate by adding the required amount of solid ammonium sulfate slowly and stirring for 30 min. The precipitated protein was dissolved in and dialyzed against dialysis buffer II overnight, and then if required, centrifuged to remove any particulate matter. The sample was loaded onto a previously equilibrated DEAE-Bio-gel column (for example, 2.5×20 cm; 100 ml bed volume for 2 L of equivalent cell culture medium volume). After washing the column with 4 bed volumes of the dialysis buffer II, protein was eluted with a linear gradient of 3.5 bed volumes of 0-800 mM NaCl (dialysis buffer II changed over to eluting buffer). The fractions were checked using 15% SDS-PAGE and the recombinant human cardiac troponin T fractions pooled then dialyzed against storage buffer. The dialysate was then centrifuged at 20,000 g for 20 min and the supernatant filtered through two layers of Miracloth.

In order to remove endotoxin from the protein preparation to produce an antigen, following Triton X-114 phase separation procedures are performed. Triton X-114 (100%) was added at 1 ml per 100 ml of the supernatant and stirred at 4° C. for 30 min. The sample was incubated at 37° C. for 10 min., then centrifuged at 20,000 g for 5 min at 30° C. using 40-ml centrifuge tubes. After removing the upper aqueous phase with care so as not to aspirate the detergent phase, the aqueous phases were combined. The addition and centrifugation steps with Triton X-114 were repeated two more times. The aqueous phase was dialyzed overnight against the storage buffer (100 ml sample per liter of the final buffer).

Final determination of protein concentration was performed using the Bradford Assay, with BSA as the standard, and final purity using a 15% SDS-PAGE. Recombinant human cardiac troponin T was stored at -20° C.

EXAMPLE 3 Expression and purification of troponin C

Human cardiac troponin C cDNA was cloned using the polymerase chain reaction (PCR) using primers designed from the published cardiac troponin C cDNA sequence (GenBank AC: X07897). The cDNA sequence was confirmed by DNA sequencing and cloned into plasmid pET21(Novagen). E. coli BL21(DE3) cells (Novagen) were transformed with the resulting construct and protein expression analyzed by SDS-PAGE.

For purification, reagents included lysis buffer (50 mM Tris-HCl, 1 mM EDTA, 20 mM 2-mercaptoethanol, 100 mM sodium chloride, pH 8.0); and storage buffer (50 mM Tris-HCl, 200 mM sodium chloride, 1mM CaCl2, pH 7.5).

Purification of recombinant human cardiac troponin C was accomplished as follows. Recombinant bacterial cells were collected by centrifugation of the cell culture. Cells were resuspend in lysis buffer at the ratio of 50 ml lysis buffer per liter equivalent of cell culture medium. Lysozyme (10 mg/ml) was added at 1 ml per liter of culture medium. The resulting mixture was then shaken at room temperature for 30 minutes. Triton X-100 (10%) was added at 5 ml per liter of original medium, followed by PMSF (0.1M) at 0.5 ml per liter of cell culture. The suspension was incubated at 37° C. for 15 minutes. The vessel containing the suspension was submerged into dry ice/ethanol to rapidly freeze the cell suspension. The frozen suspension was then thawed in a 37° C. water bath. The thawed cell suspension was sonicated using a Vibra Cell™ sonicator at 2° C.-8° C. to shear the DNA. The suspension was centrifuged at 20,000 g for 20 min at 2° C.-8° C. and the pellet was discarded. The supernatant was dialyzed in storage buffer overnight, and if required, the dialysate was centrifuged to remove any particular matter.

The supernatant was treated with 70% ammonium sulfate by adding the required amount of solid ammonium sulfate slowly and stirring for 30 min. After centrifugation, the troponin C in the supernatant was dialyzed against storage buffer overnight, then centrifuged if necessary to remove any particulate matter. The sample was concentrated in an Amicon filter to 10 ml per liter equivalent cell culture column, then was loaded on pre-equilibrated Bio-gel P60 size exclusion column. The protein was eluted with storage buffer. Fractions were checked by 15% SDS-PAGE, and the fractions containing recombinant human cardiac troponin C pooled.

In order to remove endotoxin from the protein preparation, following Triton X-114 phase separation procedures are performed. Triton X-114 (100%) was added at 1 ml per 100 ml of eluate, and the mixture was stirred at 4° C. for 30 min. Then incubated at 37° C. for 10 min. The mixture was then centrifuged at 20,000 g for 5 min. Using 40-ml centrifuge tubes, and the upper phase aspirated carefully to avoid taking the detergent phase. Aqueous phases were combined, and the detergent step repeated twice more. The aqueous phase was dialyzed against dialysis buffer overnight.

Protein concentration was determined using the Bradford Assay, with BSA as the standard. The recombinant human cardiac troponin C was lyophilized and stored at -20° C.

EXAMPLE 4 Post-purification folding of three subunits into a troponin C-I-T complex

The following reagents were used: 1.0 mg/ml recombinant human cardiac troponin I, 1.0 mg/ml recombinant human cardiac troponin C, and 1.0 mg/ml recombinant human cardiac troponin T, purified as described in the above examples; 2% sodium dodecyl sulfate; 400 mM CaCl₂ ; 400 mM MgCl2; final buffer (20 mM Tris-HCl, 500 mM NaCl, 60 mM 2-mercaptoethanol, pH 7.5); storage buffer (10 mM Phosphate buffered saline, pH 6.8, 3% normal bovine serum, 4 mM CaCl₂, 4 mM MgCl₂, protease inhibitors, and 0.05% thimerosal).

The following procedure was employed. Ten μl of 1.0 mg/ml recombinant troponin I and 10 μl of 2% SDS are combined in a microcentrifuge tube. The microcentrifuge tube was placed in a boiling water bath for 5 min., then cooled at room temperature for 5 min. Ten μl of 1.0 mg/ml recombinant troponin C, 10 μl of 1.0 mg/ml recombinant troponin T, 10 μl of 400 mM CaCl₂, and 10 μl of 400 mM MgCl₂ were added. The mixture was shaken at room temperature for 5 min., then 940 μl of the final buffer was added and the mixture shaken for an additional 25 minutes at room temperature. The above mixture was diluted into the storage buffer at 0.5 ml per 100 ml. The complex was stored at 4° C. or lyophilized.

This protocol can be scaled up proportionally to prepare larger batches of recombinant troponin complex.

EXAMPLE 5 Comparison of recombinant troponin complex stability to that of the tissue-derived complex

The stability of the recombinant cardiac troponin complex produced by the above procedures was compared directly to that of a human cardiac troponin complex prepared from human heart tissue in accordance with the protocol described in EP 0 743 522. The lyophilized powder form of the recombinant troponin complex was rehydrated and the stability monitored after reconstitution. Day 0 corresponds to the day of rehydration. After rehydration, the samples were stored at 4° C. and aliquots taken for assay at the indicated times. The initial (time 0) concentrations of each preparation are arbitrary and are not meant for comparison across experiments, but only with regard to stability over time. The data, expressed in ng/ml as measured by the Stratus(R) troponin I assay, are a follows:

                  TABLE 1                                                          ______________________________________                                                 Storage   Time Duration (days)                                         Calibrators                                                                              Conditions  0      2    7    14   28                                 ______________________________________                                         Recombinant                                                                              4 C.        14.9   15.12                                                                               16.0 16.5 15.0                               human cardiac                                                                  troponin complex                                                               in accordance                                                                  with the present                                                               invention                                                                                lyophilized 10.7   N/a  10.3 11.1 10.4                                         powder after                                                                   reconstitution and                                                             stored at 4 C.                                                       Cardiac tissue                                                                           4 C.        24.4   23.4 21.5 22.6 20.0                               troponin complex                                                               prepared                                                                       according to                                                                   EP 0 743 522                                                                             lyophilized 22.4   N/a  19.4 19.5 18.5                                         powder after                                                                   reconstitution and                                                             stored at 4 C.                                                       ______________________________________                                    

As is apparent from the data presented in Table 1, the recombinant troponin complex prepared by the method of the present invention maintained its detectability by Stratus(R) assay for 28 days at 4° C., whereas the tissue-derived human cardiac troponin complex suffered a 17-18% loss of detectability during 28 days of storage.

EXAMPLE 6 Comparison of troponin concentration determinations among various troponin I assays

Two commercial troponin I calibrators as well as the recombinant cardiac troponin complex prepared as described above were evaluated with two commercial troponin I assays and by ELISA. The concentration of recombinant human cardiac troponin I was based on protein mass as measured by Bradford using BSA as the standard. The results are presented in the following table.

                  TABLE 2                                                          ______________________________________                                                 Stated                                                                         concentration                                                                           Assay results (ng/ml)                                         Troponin source                                                                          (ng/ml)    ELISA    Stratus (R)                                                                           Access (R)                                ______________________________________                                         Procedure 25.00      26.98    21.65  22.45                                     described in the                                                               present invention                                                              Stratus (R)                                                                              27.2       0        27.9   0                                         troponin I                                                                     calibrator                                                                     Access (R)                                                                               23.34      n/a      39.5   27.4                                      troponin I                                                                     calibrator                                                                     ______________________________________                                    

As is apparent from the data presented in Table 2, each troponin source was detected near its stated concentration by the assay manufactured by that troponin source; however, in both cases the commercially-available troponin I failed to assay consistently across all assays. The troponin complex of the present invention was the only preparation measurable within reasonable limits across the assays.

Similar studies with the individual subunits of the novel complex show their improved stability and utility as standards or calibrators.

EXAMPLE 7 Preparation of a complex of troponin I and troponin C

The following reagents were used: 1.0 mg/ml recombinant human cardiac troponin I and 1.0 mg/ml recombinant human cardiac troponin C prepared and purified as described in the above examples, 10M urea in distilled water, 400 mM CaCl₂, and 400 mM MgCl₂. The Final Buffer contained 200 mM Tris-HCl, 500 mM NaCl, and 60 mM β-mercaptoethanol, pH 7.5.

The following procedure was employed. Ten μl of 1.0 mg/ml troponin I and 20 μl of 10M urea were combined in a microcentrifuge tube. The tube was shaken gently for 5 minutes at room temperature. Ten μl of 1.0 mg/ml troponin C, 10 μl of 400 mM CaCl₂, and 10 μl of 400 mM MgCl₂ were then added to the tube, which was then shaken gently for 5 minutes at room temperature, after which 940 μl of Final Buffer was added and the tube was gently shaken at room temperature for 25 minutes. This mixture can be stored at -20° C. or lyophilized.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 6                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 651 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        ATGGCTAGCATGGGATCTATGGCAGACGGTTCCAGCGATGCGGCTAGGGAACCTCGCCCT60                 GCACCAGCCCCAATCAGACGCCGCTCCTCCAACTACCGCGCTTATGCCACGGAGCCGCAC120                GCCAAGAAAAAATCTAAGATCTCCGCCTCGAGAAAATTGCAGCTGAAGACTCTGCTGCTG180                CAGATTGCAAAGCAAGAGCTGGAGCGAGAGGCGGAGGAGCGGCGCGGAGAGAAGGGGCGC240                GCTCTGAGCACCCGCTGCCAGCCGCTGGAGTTGGCCGGGCTGGGCTTCGCGGAGCTGCAG300                GACTTGTGCCGACAGCTCCACGCCCGTGTGGACAAGGTGGATGAAGAGAGATACGACATA360                GAGGCAAAAGTCACCAAGAACATCACGGAGATTGCAGATCTGACTCAGAAGATCTTTGAC420                CTTCGAGGCAAGTTTAAGCGGCCCACCCTGCGGAGAGTGAGGATCTCTGCAGATGCCATG480                ATGCAGGCGCTGCTGGGGGCCCGGGCTAAGGAGTCCCTGGACCTGCGGGCCCACCTCAAG540                CAGGTGAAGAAGGAGGACACCGAGAAGGAAAACCGGGAGGTGGGAGACTGGCGCAAGAAC600                ATCGATGCACTGAGTGGAATGGAGGGCCGCAAGAAAAAGTTTGAGAGCTGA651                         (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 867 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        ATGTCTGACATAGAAGAGGTGGTGGAAGAGTACGAGGAGGAGGAGCAGGAAGAAGCAGCT60                 GTTGAAGAGCAGGAGGAGGCAGCGGAAGAGGATGCTGAAGCAGAGGCTGAGACCGAGGAG120                ACCAGGGCAGAAGAAGATGAAGAAGAAGAGGAAGCAAAGGAGGCTGAAGATGGCCCAATG180                GAGGAGTCCAAACCAAAGCCCAGGTCGTTCATGCCCAACTTGGTGCCTCCCAAGATCCCC240                GATGGAGAGAGAGTGGACTTTGATGACATCCACCGGAAGCGCATGGAGAAGGACCTGAAT300                GAGTTGCAGGCGCTGATTGAGGCTCACTTTGAGAACAGGAAGAAAGAGGAGGAGGAGCTC360                GTTTCTCTCAAAGACAGGATCGAGAGACGTCGGGCAGAGCGGGCCGAGCAGCAGCGCATC420                CGGAATGAGCGGGAGAAGGAGCGGCAGAACCGCCTGGCTGAAGAGAGGGCTCGACGAGAG480                GAGGAGGAGAACCGTCGTAAGGCTGAGGATGAGGCCCGGAAGAAGAAGGCTTTGTCCAAC540                ATGATGCATTTTGGGGGTTACATCCAGAAGCAGGCCCAGACAGAGCGGAAAAGTGGGAAG600                AGGCAGACTGAGCGGGAAAAGAAGAAGAAGATTCTGGCTGAGCGTCGTAAGGTGCTGGCC660                ATTGACCACCTGAATGAAGATCAGCTGAGGGAGAAGGCCAAGGAGCTGTGGCAGAGCATC720                TATAACTTGGAGGCAGAGAAGTTCGACCTGCAGGAGAAGTTCAAGCAGCAGAAATATGAG780                ATCAATGTTCTCCGAAACAGGATCAACGATAACCAGAAAGTCTCCAAGACCCGCGGGAAG840                GCTAAAGTCACCGGGCGCTGGAAATAG867                                                 (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 651 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (A) DESCRIPTION: native form                                                   (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        ATGGCTAGCATGGGATCTATGGCGGATGGGAGCAGCGATGCGGCTAGGGAACCTCGCCCT60                 GCACCAGCCCCAATCAGACGCCGCTCCTCCAACTACCGCGCTTATGCCACGGAGCCGCAC120                GCCAAGAAAAAATCTAAGATCTCCGCCTCGAGAAAATTGCAGCTGAAGACTCTGCTGCTG180                CAGATTGCAAAGCAAGAGCTGGAGCGAGAGGCGGAGGAGCGGCGCGGAGAGAAGGGGCGC240                GCTCTGAGCACCCGCTGCCAGCCGCTGGAGTTGGCCGGGCTGGGCTTCGCGGAGCTGCAG300                GACTTGTGCCGACAGCTCCACGCCCGTGTGGACAAGGTGGATGAAGAGAGATACGACATA360                GAGGCAAAAGTCACCAAGAACATCACGGAGATTGCAGATCTGACTCAGAAGATCTTTGAC420                CTTCGAGGCAAGTTTAAGCGGCCCACCCTGCGGAGAGTGAGGATCTCTGCAGATGCCATG480                ATGCAGGCGCTGCTGGGGGCCCGGGCTAAGGAGTCCCTGGACCTGCGGGCCCACCTCAAG540                CAGGTGAAGAAGGAGGACACCGAGAAGGAAAACCGGGAGGTGGGAGACTGGCGCAAGAAC600                ATCGATGCACTGAGTGGAATGGAGGGCCGCAAGAAAAAGTTTGAGAGCTGA651                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 867 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (A) DESCRIPTION: native form                                                   (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        ATGTCTGACATAGAAGAGGTGGTGGAAGAGTACGAGGAGGAGGAGCAGGAAGAAGCAGCT60                 GTTGAAGAGCAGGAGGAGGCAGCGGAAGAGGATGCTGAAGCAGAGGCTGAGACCGAGGAG120                ACCAGGGCAGAAGAAGATGAAGAAGAAGAGGAAGCAAAGGAGGCTGAAGATGGCCCAATG180                GAGGAGTCCAAACCAAAGCCCAGGTCGTTCATGCCCAACTTGGTGCCTCCCAAGATCCCC240                GATGGAGAGAGAGTGGACTTTGATGACATCCACCGGAAGCGCATGGAGAAGGACCTGAAT300                GAGTTGCAGGCGCTGATTGAGGCTCACTTTGAGAACAGGAAGAAAGAGGAGGAGGAGCTC360                GTTTCTCTCAAAGACAGGATCGAGAGACGTCGGGCAGAGCGGGCCGAGCAGCAGCGCATC420                CGGAATGAGCGGGAGAAGGAGCGGCAGAACCGCCTGGCTGAAGAGAGGGCTCGACGAGAG480                GAGGAGGAGAACAGGAGGAAGGCTGAGGATGAGGCCCGGAAGAAGAAGGCTTTGTCCAAC540                ATGATGCATTTTGGGGGTTACATCCAGAAGCAGGCCCAGACAGAGCGGAAAAGTGGGAAG600                AGGCAGACTGAGCGGGAAAAGAAGAAGAAGATTCTGGCTGAGAGGAGGAAGGTGCTGGCC660                ATTGACCACCTGAATGAAGATCAGCTGAGGGAGAAGGCCAAGGAGCTGTGGCAGAGCATC720                TATAACTTGGAGGCAGAGAAGTTCGACCTGCAGGAGAAGTTCAAGCAGCAGAAATATGAG780                ATCAATGTTCTCCGAAACAGGATCAACGATAACCAGAAAGTCTCCAAGACCCGCGGGAAG840                GCTAAAGTCACCGGGCGCTGGAAATAG867                                                 (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 216 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        MetAlaSerMetGlySerMetAlaAspGlySerSerAspAlaAlaArg                               151015                                                                         GluProArgProAlaProAlaProIleArgArgArgSerSerAsnTyr                               202530                                                                         ArgAlaTyrAlaThrGluProHisAlaLysLysLysSerLysIleSer                               354045                                                                         AlaSerArgLysLeuGlnLeuLysThrLeuLeuLeuGlnIleAlaLys                               505560                                                                         GlnGluLeuGluArgGluAlaGluGluArgArgGlyGluLysGlyArg                               65707580                                                                       AlaLeuSerThrArgCysGlnProLeuGluLeuAlaGlyLeuGlyPhe                               859095                                                                         AlaGluLeuGlnAspLeuCysArgGlnLeuHisAlaArgValAspLys                               100105110                                                                      ValAspGluGluArgTyrAspIleGluAlaLysValThrLysAsnIle                               115120125                                                                      ThrGluIleAlaAspLeuThrGlnLysIlePheAspLeuArgGlyLys                               130135140                                                                      PheLysArgProThrLeuArgArgValArgIleSerAlaAspAlaMet                               145150155160                                                                   MetGlnAlaLeuLeuGlyAlaArgAlaLysGluSerLeuAspLeuArg                               165170175                                                                      AlaHisLeuLysGlnValLysLysGluAspThrGluLysGluAsnArg                               180185190                                                                      GluValGlyAspTrpArgLysAsnIleAspAlaLeuSerGlyMetGlu                               195200205                                                                      GlyArgLysLysLysPheGluSer                                                       210215                                                                         (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 288 amino acids                                                    (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        MetSerAspIleGluGluValValGluGluTyrGluGluGluGluGln                               151015                                                                         GluGluAlaAlaValGluGluGlnGluGluAlaAlaGluGluAspAla                               202530                                                                         GluAlaGluAlaGluThrGluGluThrArgAlaGluGluAspGluGlu                               354045                                                                         GluGluGluAlaLysGluAlaGluAspGlyProMetGluGluSerLys                               505560                                                                         ProLysProArgSerPheMetProAsnLeuValProProLysIlePro                               65707580                                                                       AspGlyGluArgValAspPheAspAspIleHisArgLysArgMetGlu                               859095                                                                         LysAspLeuAsnGluLeuGlnAlaLeuIleGluAlaHisPheGluAsn                               100105110                                                                      ArgLysLysGluGluGluGluLeuValSerLeuLysAspArgIleGlu                               115120125                                                                      ArgArgArgAlaGluArgAlaGluGlnGlnArgIleArgAsnGluArg                               130135140                                                                      GluLysGluArgGlnAsnArgLeuAlaGluGluArgAlaArgArgGlu                               145150155160                                                                   GluGluGluAsnArgArgLysAlaGluAspGluAlaArgLysLysLys                               165170175                                                                      AlaLeuSerAsnMetMetHisPheGlyGlyTyrIleGlnLysGlnAla                               180185190                                                                      GlnThrGluArgLysSerGlyLysArgGlnThrGluArgGluLysLys                               195200205                                                                      LysLysIleLeuAlaGluArgArgLysValLeuAlaIleAspHisLeu                               210215220                                                                      AsnGluAspGlnLeuArgGluLysAlaLysGluLeuTrpGlnSerIle                               225230235240                                                                   TyrAsnLeuGluAlaGluLysPheAspLeuGlnGluLysPheLysGln                               245250255                                                                      GlnLysTyrGluIleAsnValLeuArgAsnArgIleAsnAspAsnGln                               260265270                                                                      LysValSerLysThrArgGlyLysAlaLysValThrGlyArgTrpLys                               275280285                                                                      __________________________________________________________________________ 

What is claimed is:
 1. A recombinant human cardiac troponin I-troponin T-troponin C complex comprising a modified recombinant human cardiac troponin I, recombinant human cardiac troponin T and recombinant human cardiac troponin C, wherein the expression in E. coli of said modified recombinant troponin I is increased compared to that of a recombinant troponin I having a native protein sequence and wherein said modified recombinant troponin I consists of the native troponin I protein sequence with an N-terminal extension, said extension consisting of about 5 to about 8 amino acid residues including an N-terminal methionine.
 2. The complex of claim 1 wherein the recombinant troponin I has a sequence identified as (SEQ ID NO:5).
 3. A purified recombinant human cardiac troponin I-troponin T-troponin C complex comprising a modified recombinant human cardiac troponin I, human cardiac troponin T and human cardiac troponin C, wherein the expression in E. coli of said modified recombinant troponin I is increased compared to that of a recombinant troponin I having a native protein sequence and wherein said modified recombinant troponin I consists of the native troponin I protein sequence with an N-terminal extension, said extension consisting of about 5 to about 8 amino acid residues including an N-terminal methionine.
 4. The complex of claim 3 in which the recombinant troponin I has a sequence identified as (SEQ ID NO:5).
 5. A modified recombinant human cardiac troponin I wherein the expression in E. coli of said modified recombinant troponin I is increased compared to that of a recombinant troponin I having a native protein sequence and wherein said modified recombinant troponin I consists of the native troponin I protein sequence with an N-terminal extension, said extension consisting of about 5 to about 8 amino acid residues including an N-terminal methionine.
 6. The modified recombinant human cardiac troponin I of claim 5 having the amino acid sequence set forth in (SEQ ID NO:5).
 7. A recombinant human cardiac troponin I-troponin C complex comprising a modified recombinant human cardiac troponin I, and recombinant human cardiac troponin C, wherein the expression in E. coli of said modified recombinant troponin I is increased compared to that of a recombinant troponin I having a native protein sequence and wherein said modified recombinant troponin I consists of the native troponin I protein sequence with an N-terminal extension, said extension consisting of about 5 to about 8 amino acid residues including an N-terminal methionine.
 8. The recombinant human cardiac troponin complex of claim 7 wherein the recombinant troponin I has a sequence identified as (SEQ ID NO:5).
 9. A purified recombinant human cardiac troponin I-troponin C complex comprising a modified recombinant human cardiac troponin I and human cardiac troponin C, wherein the expression in E. coli of said modified recombinant troponin I is increased compared to that of a recombinant troponin I having a native protein sequence and wherein said modified recombinant troponin I consists of the native troponin I protein sequence with an N-terminal extension, said extension consisting of about 5 to about 8 amino acid residues including an N-terminal methionine.
 10. The complex of claim 9 which the recombinant troponin I has a sequence identified as (SEQ ID NO:5). 